Shosaku Hattori

Accelerated evolution of crotalinae snake venom gland serine proteases

Eight cDNAs encoding serine proteases isolated from Trimeresurus flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein‐coding regions to cause amino acid changes. Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been previously discovered in crotalinae snake venom gland phospholipase A2 (PLA2) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.

Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes.

Three Trimeresurus okinavensis (To; himehabu snake, Crotalinae) venom gland phospholipase A2 (PLA2) isozymeencoding genes, gPLA2-o1, gPLA2-o2 and gPLA2-o3, were isolated from its genomic DNA library. The nucleotide (nt) sequence analysis revealed that two of the three genes (gPLA2-o2 and gPLA2-o3) occasionally have been converted to inactivated genes by introduction of one base insertion or substitution. It was confirmed from Southern blot analysis that the To haploid genome contains only three venom gland PLA2 isozyme genes herein isolated. Comparison of these genes showed that nonsynonymous nt substitutions have occurred more frequently than synonymous nt substitutions in the protein-coding regions, except for the signal-peptide coding domain, implying that To venom gland PLA2 isozyme genes have evolved via accelerated evolution. Such an evolutionary feature of To venom gland PLA2 isozyme genes proves the general universality of accelerated evolution previously drawn for venom gland PLA2 isozyme genes of other crotalinae snakes. The variability in the mature protein-coding regions of three To venom gland PLA2 isozyme genes appears to have been brought about by natural selection for point mutations.

Interisland Evolution of Trimeresurus flavoviridis Venom Phospholipase A2 Isozymes

Abstract Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A2 (PLA2), named PL-Y, was isolated from Okinawa T. flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA2 from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA2, named PLA-B′, which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp49]PLA2 isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima), and PLA-B′ (Amami-Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the β1-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different environments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA2, named PLA2, ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA2 gene is a prototype among T. flavoviridis venom PLA2 isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA2 isozymes are clearly separated into three groups, PLA2 type, basic [Asp49]PLA2 type, and [Lys49]PLA2 type. Basic [Asp49]PLA2-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys49]PLA2 type.

Flavoxobin, a serine protease from Trimeresurus flavoviridis (habu snake) venom, independently cleaves Arg726-Ser727 of human C3 and acts as a novel, heterologous C3 convertase

We have recently shown that crude Trimeresurus flavoviridis (habu snake) venom has a strong capability for activating the human alternative complement system. To identify the active component, the crude venom was fractionated and purified by serial chromatography using Sephadex G‐100, CM‐cellulose C‐52, diethylaminoethyl‐Toyopearl 650M, and Butyl‐Toyopearl, and the active fractions were evaluated by the C3a‐releasing and soluble membrane attack complex‐forming activities. Two peak fractions with the highest activities were detected after gel filtration and ion exchange chromatography, and the first fraction was purified to homogeneity. The homogeneous protein was examined for its N‐terminal amino acid sequence by Edman degradation. The determined sequence of 25 amino acids completely coincided with that of a previously reported serine protease with coagulant activity, flavoxobin, purified from the same snake venom. To elucidate the molecular mechanism of the complement activation, the reactive products of the mixture of the purified human C3 and flavoxobin were examined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The digesting pattern revealed that flavoxobin cleaves the α chain of the C3 molecule into two fragments. The N‐terminal amino acid sequences for the remnant fragments of C3 disclosed that flavoxobin severs the human C3 at the Arg726‐Ser727 site to form C3b and C3a the way C3bBb, the human alternative C3 convertase, does. In conclusion, flavoxobin acts as a novel, heterologous C3 convertase that independently cleaves human C3 and kick‐starts the complement cascade.

Chimeric VEGF-ENZ7/PlGF Specifically Binding to VEGFR-2 Accelerates Skin Wound Healing via Enhancement of Neovascularization

Objective—VEGF-ENZ7/PlGF molecules composed of Orf virus-derived VEGF-ENZ7 and human PlGF1 were previously proven to be potent angiogenic factors stimulating angiogenesis without significant enhancement of vascular leakage and inflammation in vivo. For its future clinical application, there is a pressing need to better understand the beneficial effects of VEGF-ENZ7/PlGF during wound healing in adulthood. Methods and Results—In this study, several angiogenic factors were administrated to skin punched wounds of both wild-type and diabetic mice. The treatment with VEGF-ENZ7/PlGF accelerated wound closure accompanied with enhanced angiogenesis, the process was occurring slightly faster than that in VEGF-A164 group. Moreover, the macrophage infiltration and lymphangiogenesis level in healed wounds were strikingly lower in VEGF-ENZ7/PlGF group than VEGF-A164 group, suggesting that the increased inflammation was the key issue preventing speedy wound healing of VEGF-A164–treated skin. Considering clinical safety, we further examined the antigenicity of chimeric VEGF-ENZ7/PlGF. Compared with the original VEGF-ENZ7, the immunogenicity of VEGF-ENZ7/PlGF molecules was markedly decreased in mice and squirrel monkeys with the increase of PlGF1 humanized ratio. Conclusion—These results indicate that VEGF-ENZ7/PlGF molecules are superior to VEGF-A for the acceleration of either normal or delayed skin wound healing and might be regarded as potential drugs in therapeutic angiogenesis.

Interisland mutation of a novel phospholipase A2 from Trimeresurus flavoviridis venom and evolution of Crotalinae group II phospholipases A2.

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A2 (PLA2). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA2, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD50 for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA2’s based on the amino acid sequences revealed that neurotoxic PLA2’s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA2 groups such as PLA2 type, basic [Asp49]PLA2 type, and [Lys49]PLA2 type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA2’s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA2‘s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA2’s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation.

Structural elements of Trimeresurus flavoviridis serum inhibitors for recognition of its venom phospholipase A2 isozymes

Five inhibitors (PLI‐I–V) against Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) venom phospholipase A2 (PLA2) isozymes have been isolated from its serum. PLI‐I, which is composed of two repeated three‐finger motifs, and PLI‐IV and PLI‐V, which contain a sequence similar to the carbohydrate recognition domain (CRD) of C‐type lectins, were expressed in the forms fused with glutathione S‐transferase (GST). The resulting GST‐PLIs showed ability to bind to three Tf venom PLA2 isozymes. The binding study with the truncated forms indicated that one of two three‐finger motifs of PLI‐I was able to bind to PLA2 isozymes. The N‐terminal 37‐amino acid fragment and the CRD‐like domain of PLI‐IV and PLI‐V were bound to PLA2 isozymes. On the other hand, their C‐terminal 12‐amino acid segment also associated with PLA2 isozymes. When either of two units of a hydrophobic tripeptide in this sequence was replaced by trialanine, the binding was completely abolished, indicating that the C‐terminal hydrophobic cores of PLI‐IV and PLI‐V were critically responsible for the binding to venom PLA2 isozymes.

Structures of genes encoding phospholipase A2 inhibitors from the serum of Trimeresurus flavoviridis snake

Inhibitors (PLIs) against snake venom gland phospholipases A2 (PLA2s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA2 isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region.

Structures of genes encoding TATA ☐-binding proteins from trimeresurus gramineus and t. flavoviridis snakes

A cDNA encoding the Trimeresurus gramineus (Tg; green habu snake) TATA-box-binding protein (TgTBP) was cloned and sequenced. The cDNA encodes a 33-kDa protein with an extensive sequence similarity to those derived from other organisms, except for the N-terminal domain. Genes encoding TgTBP and Trimeresurus flavoviridis (Tf; habu snake) TBP (TfTBP) were isolated using a TgTBP cDNA and their nt sequences were determined. They are the first TBP genes entirely sequenced in higher animals. Both genes span over 15 kb and are constructed from eight exons and seven introns. Comparison of the loci of introns on the aligned amino-acid sequences of TBP from six organisms (Tg, Tf, mouse, Arabidopsis thaliana, Schizosaccharomyces pombe and Acanthamoeba castellanii) indicated that there are three highly conserved loci in the C-terminal domain.

Measles virus selectively blind to signaling lymphocyte activation molecule as a novel oncolytic virus for breast cancer treatment

Oncolytic viruses hold much promise as novel therapeutic agents that can be combined with conventional therapeutic modalities. Measles virus (MV) is known to enter cells using the signaling lymphocyte activation molecule (SLAM), which is expressed on cells of the immune system. Although human breast cancer cell lines do not express SLAM, we found that a wild-type MV (HL strain) efficiently infected various breast cancer cell lines, causing cell death. Based on this finding, we used reverse genetics to generate a recombinant MV selectively unable to use SLAM (rMV-SLAMblind). The rMV-SLAMblind lacked infectivity for SLAM-positive lymphoid cells, while retaining oncolytic activity against breast cancer cells. We showed that, unlike the MV vaccine strains, rMV-SLAMblind used PVRL4 (polio virus receptor-related 4) as a receptor to infect breast cancer cells and not the ubiquitously expressed CD46. Consistent with this, rMV-SLAMblind infected CD46-positive primary normal human cells at a much-reduced level, whereas a vaccine strain of the Edmonston lineage (rMV-Edmonston) efficiently infected and killed them. The rMV-SLAMblind showed antitumor activity against human breast cancer xenografts in immunodeficient mice. The oncolytic activity of rMV-SLAMblind was significantly greater than that of rMV-Edmonston. To assess the in vivo safety, three monkeys seronegative for MV were inoculated with rMV-SLAMblind, and no clinical symptoms were documented. On the basis of these results, rMV-SLAMblind could be a promising candidate as a novel oncolytic virus for breast cancer treatment.